The termination of neurotransmitter action is determined by a number of factors, including their reuptake into nerve terminals by monoamine transporters, their dilution by diffusion out of the synaptic cleft, and their metabolism by Monoamine Oxidase. Specific monoamine transporters located in the neuronal plasma membrane terminate the action of neurotransmitters by transporting them back into presynaptic terminals. Once inside the presynaptic terminal, vesicular monoamine transporters mediate their filling into secretory vesicles. All characterized monoaminergic cells utilize the vesicular monoamine transporter (VMAT) to accumulate monoamines from the cytoplasm into vesicles. These VMATs are polytopic membrane proteins, which act as electrogenic antiporters (exchangers) of protons and monoamines utilizing an acidic and positively polarized granule matrix.
The monoamine transporters of synapses formed by the midbrain dopamine projections are involved in voluntary motor control, reward and learning, and are the primary target of drugs of abuse including amphetamine, nicotine, cocaine as well as therapeutic agents that are used to treat mood disorders. Neuronal death in the substantia nigra is the cause of Alzheimer's disease and a decreased density of dopamine monoamine transporter has been found in Parkinson's, Wilson's, and Lesch-Nyhan's disease, while a decrease in serotonin monoamine transporter level is found in patients suffering from major depression and aggressive behavior.
Some evidence suggests that monoamine transporters recognize compounds other than neurotransmitters as substrates (1). This gave rise to the “false neurotransmitter hypothesis”—that different monoamines are transported into the same vesicle, resulting in the accumulation and release of so-called “false neurotransmitters”. If, and to what extent, this apparent promiscuity is a fundamental biochemical property of the monoamine transporters still remains an unanswered question today.
The currently available molecular imaging tools do not enable monitoring of vesicle loading and release with spatial resolution of single terminals. The fluorescent amine acridine orange (21) accumulates in all acidic neuronal compartments, while fluorescent ASP+ stains mitochondria and cytosol but not vesicles (4,5,6,22). Currently, the dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter (SERT) activities are measured by ASP+, a fluorescent analog of MPP+. However, due to ASP+ and acridine orange's shortcomings mentioned above, they cannot be readily used to monitor transmission in living cells or brain slices.
Moreover, current approaches for direct measurement of monoamine release rely on microdialysis and electrochemical methods. Although electrochemical detection of dopamine (DA) release with cyclic voltammetry and amperometry has provided excellent temporal resolution, (38) these methods provide poor spatial resolution in brain tissue as they sample release and uptake of hundreds to thousands of DA terminals.
Herein is described a novel optical approach based on “optical fluorescent transmitters” or “fluorescent false neurotransmitters” (“FFNs”) that act as optical tracers, providing the first direct means to directly visualize neurotransmitter uptake, redistribution, and release from individual dopamine terminals. FFNs were designed by targeting the synaptic vesicular monoamine transporter (VMAT2) that transports dopamine and other aminergic neurotransmitters from the cytoplasm into synaptic vesicles. Like dopamine, these probes selectively accumulate in dopamine and other aminergic neurotransmitters from cytoplasm into vesicles. Like dopamine, these probes selectively accumulate in dopamine terminals in the brain in a manner dependent on VMAT2 function and the vesicular pH gradient, and are released upon synaptic firing.